EP0304578A1 - Peptide contenant l'antigène de surface de l'hépatite B - Google Patents

Peptide contenant l'antigène de surface de l'hépatite B Download PDF

Info

Publication number
EP0304578A1
EP0304578A1 EP88109946A EP88109946A EP0304578A1 EP 0304578 A1 EP0304578 A1 EP 0304578A1 EP 88109946 A EP88109946 A EP 88109946A EP 88109946 A EP88109946 A EP 88109946A EP 0304578 A1 EP0304578 A1 EP 0304578A1
Authority
EP
European Patent Office
Prior art keywords
peptide
hbv
dna molecule
host cell
recombinant dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP88109946A
Other languages
German (de)
English (en)
Other versions
EP0304578B1 (fr
Inventor
Hans A. Dr. Thoma
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medeva Holdings BV
Original Assignee
MCCORMICK AND JONES ENGINEERING Ltd
Mccormick & Jones Eng
Medeva Holdings BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MCCORMICK AND JONES ENGINEERING Ltd, Mccormick & Jones Eng, Medeva Holdings BV filed Critical MCCORMICK AND JONES ENGINEERING Ltd
Priority to EP00121167A priority Critical patent/EP1088830A3/fr
Priority to EP88109946A priority patent/EP0304578B1/fr
Publication of EP0304578A1 publication Critical patent/EP0304578A1/fr
Application granted granted Critical
Publication of EP0304578B1 publication Critical patent/EP0304578B1/fr
Priority to LU90911C priority patent/LU90911I2/fr
Priority to NL300090C priority patent/NL300090I2/nl
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to Hepatitis B surface antigen ("HBs antigen” or "HBsAG”) particles which are composed of polypeptides prepared by recombinant DNA processes, DNA sequences coding for these polypeptides and cell lines for the expression of the same.
  • HBs antigen or "HBsAG”
  • the present invention relates especially to new particles having increased immunogenicity.
  • polypeptide antigens produced in vivo are heavily glycosilated (Gerlich, 1984: J. Virol.: 52 (2), 396).
  • glycosilation is not an essential process so that polypeptides produced by genetically engineered bacteria are either not glycosilated or are incompletely glycosilated.
  • polypeptides corresponding to HBsAg when expressed in bacteria, do not raise antibodies which will see HBsAg sufficiently well for an effective vaccine.
  • yeast as a eukaryotic host is capable of more complete glycosilation, polypeptides corresponding to HbsAg expressed in yeast share the same deficiency as in the case of bacterial expression. (Murray et al., 1979: Nature, 282, 575; Valenzuela et al., 1982: Nature, 298, 347; Miyanohara et al., 1983: PNAS, 80, 1).
  • the eukaryotic structural gene of the HBsAg is in most cases not efficiently transcribed. Furthermore the structure and function of the eukaryotic HBsAg gene product may be dependent on the additional post-translational processes of the linkage of disulfide bonds which can not be accomplished by the bacterial host.
  • the expressed polypeptide is rarely secreted from the bacterial host cells. They must be lysed to harvest the expressed polypeptide. During the purification process bacterial wall components may contaminate the polypeptide and cause serious allergic reactions or lead to anaphylactic shock in patients.
  • eukaryotic promoters usually do not work in bacteria and must be substituted by a bacterial promoter which can result in modification of the polypeptide expressed.
  • HBV Hepatitis B virus
  • HBV protein occurs in three distinct morphologies: - the HBV-virion (Dane particle), which is through to be the infectious material, - the filaments, and - the 20 or 22 nm particles (hereinafter "20 nm particle”) which consist only of a protein envelope.
  • the most interesting form for an efficient vaccine is the 20 nm particle because 1) the coding sequences are entirely known, 2) it is completely uninfectious, and 3) it causes some useful immunogenicity in a human organism.
  • HBV particles differ in their relative amounts of the protein composition. There are three monomers called the major protein with 226 amino acids, the middle protein with 281 amino acids, and the large protein with 389 or 400 amino acids, depending on the subtype ayw and adw, respectively.
  • the large protein is encoded by the complete sequence of the pre-S1-, pre-S2- and S-regions, whereas the middle protein is derived from only the pre-S2- and S-­regions, and finally the major protein from only the S-region (Tiollais et al., 1985: Nature, 317, 489; Dubois et al., 1980: PNAS, 77, 4549; McAlzer et al., 1984: Nature, 307, 178).
  • the infectious virion of HBV contains 40-80 times more of the high molecular monomers - the pre-S1 and pre-S2 peptides - compared to the 20 nm particle. It is now known that these pre-S polypeptides may be associated with some biological and clinical implications.
  • the polyalbumin receptor on the pre-S polypeptides can bind polymerized albumins from humans and chimpanzees which are susceptible to HBV (Thung et al., 1983: Liver, 3, 290; Machida et al., 1984: Gastroenterology, 86, 910).
  • Literature data would also suggest a better protection against the infectious Dane-particle where the pre-S1 epitope is present in much higher ratio than on the envelope particles.
  • the vaccine obtained from natural sources which causes a limited immunogenic protection, contains (almost) none of the pre-S proteins; this is due to two different reasons.
  • the 20 nm particles are isolated from sera of anti-HBE positive carriers (Hevac B, HepaVac B) or are digested by proteases during the purification process. This proteolytic digestion has been shown to cut the pre-S-polypeptides leaving only the S monomers. As a result these vaccines contain none or very little pre-S polypeptides.
  • EP-A-72 318 describes the expression of HBsAg in yeast cells, which have been transformed by a vector comprising a yeast replicon, a yeast promoter and a DNA sequence coding for the S peptide.
  • Laub et al. J. Virol., Vol. 48, No. 1, pp. 271-280, 1983, disclose the construction of a vector starting from simian virus 40 into which the HBsAg including the 163 codon precursor sequence was incorporated. Laub et al. report that CV-1 cells transformed with said vector yield a better expression when the vector contains only the coding sequence for the S protein as compared to the above vector which comprises additionally also the 163 codon precursor sequence.
  • Japanese Patent Application No. J5-8194-897-A describes the expression of the entire pre-S and S peptides. Reference is also made to the expression of the adw subtype.
  • DE-OS 34 39 400 describes the expression of an immunogenic polypeptide sequence of Hepatitis B virus.
  • Said sequence represents a partial sequence of the pre-S1 polypeptide, comprises 108 or 119 codons and starts with the first starting codon of HBsAg, and terminates 281 codons in front of the stop codon.
  • EP-A-154 902 discloses a Hepatitis B vaccine which contains a peptide with an amino acid chain of at least six consecutive amino acids within the pre-S chain coding region of the envelope of Hepatitis B virus. This vaccine is free of an amino acid sequence corresponding to the naturally occurring envelope proteins of Hepatitis B virus.
  • the object of this invention is the development of protein particles which contain an amount of the pre-S polypeptide epitopes comparable to the natural composition of the surface structure of the infectious Dane particle.
  • HBsAg in mammalian cells. This requires overcoming known difficulties where expression of the desired peptide in a mammalian cell can result in: - different regulatory mechanisms for the three translational/(transcriptional) products - promoter-promoter inhibition - different strength of the start codons - not all peptides expressed.
  • HBV S peptide refers to the peptide encoded by the entire S region of the HBV genome.
  • HBV pre-S2 peptide refers to the peptide encoded by the entire pre-S2 and S regions of the HBV genome.
  • HBV pre-S1 peptide refers to the polypeptide encoded by the entire pre-S1, pre-S2 and S regions of the HBV genome.
  • epitope refers to a sequence of at least six consecutive amino acids encoded by the designated genome region (e.g., a "HBV pre-S2 epitope” refers to a sequence of at least six amino acids encoded by the pre-S2 regions of the HBV genome).
  • antigenicity means the ability to provoke an immune response (e.g., acting as a vaccine or an antigen), the ability to cause the production of antibodies (e.g. acting as an antigen) and/or the ability to interact with a cell surface receptor so as to enhance an immune response or production of antibodies (e.g., reacting with a T-cell surface receptor to enhance immune response).
  • HBV means any subtype of the virus, particularly adw, ayw, adr and ayr, described in the literature (P. Valenzuela, Nature Vol. 280, p. 815 (1979), Gerlich, EP-A-85 111 361, Neurath, EP-A-85 102 250). Examples of peptide sequences thereof, from which the epitopes of this invention can be derived, are shown in Figures XVI to XX.
  • recombinant DNA molecules which comprise a first DNA sequence and a second DNA sequence.
  • the first DNA sequence encodes for expression of an amino acid sequence a portion of which displays the antigenicity of an epitope selected from the group consisting of an HBV pre-S1 epitope and an HBV pre-S2 epitope.
  • the second DNA sequence encodes for expression of a peptide which upon secretion will form particles which are at least 10 nm in diameter. These particles are believed to be the smallest particles which will effectively form a good vaccine.
  • the peptide which upon secretion will form particles which are at least 10 nm in diameter is either HBV S peptide, HBV core antigen, polio surface antigen, Hepatitis A surface antigen, Hepatitis A core antigen, HIV surface antigen and HIV core antigen.
  • a substantial portion or all of the HBV S peptide is especially preferred as the peptide encoded by the second DNA sequence.
  • the recombinant DNA molecules encoding for the first and second DNA sequences must be (1) in the same reading frame, (2) encode for respective discrete regions of a single peptide, and (3) be operatively linked to an expression control sequence. Finally, these recombinant DNA molecules are free of DNA sequences encoding for the expression of the entire HBV pre-S1 peptide or HBV pre-S2 peptide.
  • the first DNA sequence comprises a nucleotide sequence corresponding to the nucleotide sequence of (1) the HBV pre-S1 and pre-S2 regions from which the pre-S2 start codon ATG has been deleted, (2) the HBV pre-S1 and pre-S2 regions and wherein the sequences flanking the pre-S1 ATG have been changed from the natural sequence, (3) the HBV pre-S1 and pre-S2 regions and wherein the sequences flanking the pre-S2 ATG have been changed from the natural sequence, (4) the HBV pre-S1 and pre-S2 regions and wherein the 5′ terminus of the pre-S1 region has been deleted, (5) the HBV pre-S1 and pre-S2 regions and wherein the 5′ terminus of the pre-S2 region has been deleted, (6) the HBV pre-S1 region and wherein the 3′ terminus of the pre-S1 region has been deleted, (7) the HBV pre-S2 region and wherein
  • Host cells transfected with the recombinant DNA molecules of the present invention are also disclosed.
  • transfected or transfection refer to the addition of exogenous DNA to a host cell whether by transfection, transformation or other means.
  • Host cells include any unicellular organism capable of transcribing and translating recombinant DNA molecules including without limitation mammalian cells, bacteria and yeast.
  • Host cells of the present invention may also be cotransfected with a second recombinant DNA molecule comprising a DNA sequence encoding for expression of an amino acid sequence corresponding to a substantial portion or all of the amino acid sequence of the HBV S peptide.
  • Peptides comprising a first discrete region and a second discrete region.
  • the first region displays the antigenicity of an epitope of an HBV pre-S1 epitope or an HBV pre-S2 epitope.
  • the second region correspond to a substantial portion of a peptide which upon secretion will form particles which are at least 10 nm in diameter.
  • the peptide which upon secretion will form particles which are at least 10 nm in diameter is either HBV S peptide, HBV core antigen, polio surface antigen, Hepatitis A surface antigen, Hepatitis A core antigen, HIV surface antigen and HIV core antigen.
  • a substantial portion or all of the HBV S peptide is especially preferred.
  • the first region is located closer to the N-terminus of the peptide than the second region.
  • Immunogenic particles are also disclosed which comprise a plurality of first peptide monomers.
  • Each of said first peptide monomers comprises a first discrete region and a second discrete region which can be the same as the first and second discrete regions of the peptides described above.
  • Immunogenic particles are also disclosed which further comprise a plurality of second peptide monomers and wherein the first and second peptide monomers are bound together by interactive forces between the monomers.
  • Each of said second peptide monomers comprising an amino acid sequence corresponding to a substantial portion of or all of the amino acid sequence of the HBV S peptide.
  • Immunogenic particles are also disclosed which contain substantially more than one percent, preferably more than five percent, of the pre-S1 epitope.
  • a particle "contains one percent" of a designated epitope if peptide monomers having the designated epitope constitute one percent of all protein in the particle.
  • Immunogenic particles which contain substantially more than ten percent, preferably more than fifteen percent, of the pre-S2 epitope are also disclosed.
  • Suitable carriers are known to those skilled in the art and may include simple buffer solutions.
  • compositions useful for production of antibodies comprising the above-described immunogenic particles in sufficient concentration to elicit an immune response upon administration of said preparation and a suitable carrier.
  • suitable carriers are known to those skilled in the art and may include simple buffer solutions.
  • a process for producing a transfected hose cell comprises providing host cells which have been made competent for uptake of DNA, exposing the host cells to a first preparation of DNA comprising one of the above-described recombinant DNA molecules, allowing under suitable conditions the host cells to take up DNA from the first preparation of DNA, and selecting for host cells which have taken up exogenous DNA.
  • the process may further comprise exposing the host cells to a second preparation of DNA comprising a DNA molecule encoding for a peptide including the amino acid sequence of the HBV S peptide and allowing under suitable conditions the host cells to take up DNA from the second preparation of DNA.
  • the exposure and uptake of the second preparation of DNA can be done before or after exposure to and uptake of the first DNA preparation.
  • the first DNA preparation can also include a DNA molecule encoding for a peptide including the amino acid sequence of the HBV S peptide.
  • a method for producing a peptide comprises preparing an above-described recombinant DNA molecule, transfecting a host cell with the recombinant DNA molecule, culturing the host cell under conditions allowing expression and secretion of protein by the host cell, and collecting the peptide produced as a result of expression of DNA sequences within the recombinant DNA molecule.
  • the peptide produced by such method can contain less than the entire amino acid encoded by the coding region of the recombinant DNA molecule. This may result from transcription and/or translation of only a portion of the coding region of the recombinant molecule or by deletions made in the peptide after translation.
  • a method of producing immunogenic particles comprising preparing an above-described recombinant DNA molecule, transfecting a host cell with the recombinant DNA molecule, culturing the host cell under conditions allowing expression and secretion of protein by the host cell, and allowing under suitable conditions the aggregation of peptide monomers produced as a result of expression of exogenous DNA sequences within the host cell.
  • a method of producing immunogenic particles is also disclosed which further comprises transfecting (cotransfection) the host cell with a DNA molecule encoding for a peptide including the amino acid sequence of the HBV S peptide. The cotransfection can occur before, after or simultaneous with the transfection of the above-described recombinant DNA molecule. Presence of peptides encoded by the cotransfected DNA molecule are necessary to obtain more than trace amounts of particles secreted from the host cell.
  • Methods of manufacturing a pharmaceutical preparation and a preparation useful for production of antibodies comprising preparing an above-described recombinant DNA molecule, transfecting a host cell with the recombinant DNA molecule, culturing the host cell under conditions allowing expression and secretion of protein by the host cell, allowing under suitable conditions the aggregation of peptides produced as a result of expression of DNA sequences within the host cell to form immunogenic particles, and combining the immunogenic particles with a suitable carrier such that the immunogenic particles are present in sufficient concentration to cause production of antibodies upon administration of a preparation to an individual.
  • Host cells used in these methods can also be cotransfected as previously described.
  • Preferred DNA constructs of the present invention are characterized by the presence of a selection marker selected from the group consisting of dhfr (dihydrofolate reductase), MT-neo (a neomycin resistance sequence coupled to a methallothionein and MT-ecogpt (a resistance sequence coupled to a methallothionein promoter).
  • a selection marker selected from the group consisting of dhfr (dihydrofolate reductase), MT-neo (a neomycin resistance sequence coupled to a methallothionein and MT-ecogpt (a resistance sequence coupled to a methallothionein promoter).
  • the expression rate may be further enhanced by adding to the constructs a dhfr gene as an amplification gene.
  • HBV nucleotide sequences used in certain constructs of the present invention can be formed or isolated by any means including isolation and ligation of restriction fragments and synthetic oligonucleotides. Constructs specifically described herein were formed by the ligation of synthetic oligonucleotides to a 5′ XbaI-BglII 3′ fragment from the S region of the HBV genome shown in Figure IX (hereinafter the "XbaI-BglII fragment") which is derived from a BglII-BglII HBV fragment including the entire pre-S1-pre-S2-S regions (the "BglII-BglII Fragment") The pre-S1-pre-S2-S region of the HBV genome is shown in Figure IX.
  • Oligonucleotides used in making such constructs are summarized in Table I.
  • Table I Oligonuclcotide Duplexes for Vector Construction Oligo.
  • No. Restriction Sites and Schematic Structure Function Sequence (5′-3′) (sticky ends are underlined) 1 MstII-ATG-S1-XbaI S1 (exchanged flanking sequence ATG) TCAGG AAATGGAGAACATATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGT 13 MstII-ATA-S1-XbaI S1 (exchanged flanking sequence ATA) TCAGG AAATAGAGAACATATCAGGATTCCTAGGACCCCTTCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGT 17 BglII-ATG-S2-EcoRI S2 (exchanged flanking sequence ATG) GATC TACCTG
  • oligonucleotides in Table I were combined with the XbaI-BglII fragment to produce constructs with desired features.
  • adapter oligonucleotide sequences (Table II) were used to create proper matching sticky ends on the oligonucleotides and other construct components.
  • adapter sequences may be used to combine desired oligonucleotides from Table I with the XbaI-BglII fragment, other restriction fragments, oligonucleotides and other construct components.
  • the necessary sequences of such other adapter sequences will be readily apparent to those skilled in the art from consideration of tables of restriction sites [e.g., that found at pages 121-128 of Methods in Enzymology , volume 152, "Guide to Molecular Cloning Techniques," ed. Berger and Kimmel (Academic Press 1987) which is incorporated herein in its entirety by reference] and the sequences of the various nucleotides to be combined.
  • Adapter sequences can also be used to introduce additional restriction sites into constructs of the present invention. It should be noted that adapter sequences must be selected or designed so that the proper reading frame is maintained throughout the HBV sequence.
  • Preferred gene constructs which were used to transfect host cells were prepared by recombinant DNA techniques in accordance with the present invention.
  • Preferred embodiments of constructs with an enhanced expression rate are shown in Figures I-VIII and are schematically represented by the following: pU2-structural gene pU2-structural gene-dhfr pU2-structural gene-dhfr-MT-neo pU2-structural gene-dhfr-MT-egpt pMT-structural gene-dhfr pMT-structural gene-dhfr-MT-neo pMT-structural gene-dhfr-MT-egpt pH2K-structural gene-dhfr pH2K-structural gene-MT-neo pH2K-structural gene-MT-egpt pH2K-structural gene-dhfr-MT-neo pH2K-structural gene-dhfr-MT-neo pH2K-structural gene-dhf
  • Each of the constructs shown in Figures I-VIII contain, in addition to a HBV sequence, a neomycin selection marker with the MT promoter, an ampicillin selection marker, a dhfr selection/amplification gene and a promoter for the HBV sequence.
  • the promoter for the HBV sequence is preferably the U2 promoter, the MT promoter or the H2K promoter. Isolation of fragments containing the various promoters, the selection markers and amplification gene is described below.
  • HBV sequences in the constructs of Figures I-VIII are schematically represented by a rectangular bar in each figure which indicates the oligonucleotides and/or adapter sequences from Tables I and II which were combined with the XbaI-BglII fragment. Shaded areas within the bar indicate generally regions of the entire pre-S1-pre-S2-S region which are not found in the specific construct. Oligonucleotides from Table I which can be used to construct each type of HBV sequence are indicated in the figures.
  • Figure X depicts two additional constructs for expression of peptides including sequence from the pre-S2 region under the control of the MT promoter.
  • the above-cited promoters are specially preferable when their use is coupled with a modulation method using the dhfr gene and methotrexate to enhance the expression. This is achieved when in addition to the selection marker the dhfr minigene is also introduced into the plasmid sequence. It is essential that the dhfr gene is located on the same plasmid together with the structural gene to be expressed. An enhancement of the expression rate of the structural gene can then be obtained by adding methotrexate in the micromolar concentration range. Thereby a manyfold enhancement of the expression rate is achieved.
  • Suitable cells are e.g. VERO cells (monkey kidney cell line), 3T3-cells (murine fibroblast line), C127-cells (murine fibroblast line), L-cells and CHO - cells (Chinese hamster cells, which are either positive or negative in dehydrofolate reductase).
  • HBV protein refers generically to any protein produced in accordance with the present invention which corresponds to HBsAG sequences.
  • the supernatant of HBV protein producing cultures was collected and split into portions of 2,400 ml. To each portion 144 g of PEG 6000 (Serva) were added and dissolved by stirring at room temperature for 20 minutes and was stirred for another 6 hours at 4°C. The precipitate was separated by centrifugation in 500 ml bottles in a GS 3 rotor at 9,000 rpm (15,000 x g) for 30 minutes at 10 C. The supernatant was collected and 144 g of PEG 6000 were added and dissolved as described above. The solution was stirred at 4 C for 3 hours. The precipitate from this solution was harvested as described above except that centrifugation was continued for 60 minutes.
  • HBsAg micro "sandwich” assay commercially available from Behring
  • wells were coated with anti-HBs.
  • Serum plasma or purified protein and appropriate controls were added to the wells and incubated.
  • peroxidase-labelled antibodies to HBsAg were reacted with the remaining antigenic determinants.
  • the unbound enzyme-linked antibodies are removed by washing and the enzyme activity on the solid phase is determined.
  • the enzymatically catalyzed reaction of hydrogen peroxide and chromogen was stopped by adding diluted sulfuric acid.
  • the colour intensity was proportional to the HBsAg concentration of the sample and was obtained by photometric comparison of the colour intensity of the unknown samples with the colour intensities of the accompanying negative and positive control sera.
  • the plasmid pBPV-342-12 (commercially available from ATCC) was digested with the endonucleases BglII and BamHI. Three DNA molecules were generated.
  • the fragment of interest contains the methallothionein promoter and a pBR322 sequence comprising 4.5 kb and is easily detectable from the other fragments (2.0 kb and 7.6 kb).
  • the reaction was performed in a total volume of 200 ⁇ l of reaction buffer at a final concentration of 0.5 ⁇ g/ ⁇ l DNA including 100 units of each restriction enzyme. The completion of the digestion was checked after incubation at 37°C for three hours by agarose gel electrophoresis at a 0.8% agarose gel. The reaction was stopped by adding 4 ⁇ l 0.5 M EDTA.
  • the 4.5 kb fragment was separated from the other fragments by preparative 1.2% agarose gel electrophoresis.
  • the DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer.
  • the DNA was purified by a phenol/chloroform extraction and two ethanol precipitations.
  • a 2.3 kb BglII-BglII fragment containing the HBV pre-S1,pre-S2 and S coding regions was isolated from HBV- containing DNA.
  • the 2-3kb fragment was ligated together with the 4.5 kb fragment (obtained as described in Cl) containing the methallothionein promoter.
  • the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. After the DNA up-date the bacterial cells were spread on LB agar plate containing 50 ⁇ g/ml ampicillin at volumes of 50 to 300 ⁇ l cell suspension per plate. The agar plates were incubated at 37°C overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired fragments.
  • the plasmid resulting from (3) above was digested with the endonucleases BglII and XbaI. Two molecules were expected, one 550 bp fragment and one 6.250 kb fragment which was isolated after agarose gel electrophoresis.
  • the 6.250 kb fragment was ligated together with oligomecleotide No.55 from Table I.
  • the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. Single isolated bacterial colonies were screened for the presence of the desired plasmid.
  • the new plasmid was proved by a digestion with the endonucleases EcoRI and BglII. Two molecules were expected, one 1.9 kb and one 4.450 kb.
  • the plasmid resulting from (4) above was linearized by digestion with the restriction enzyme EcoRI.
  • the reaction was performed in a total volume of 50 ⁇ l and a final concentration of 1 ⁇ g/ ⁇ l plasmid DNA. 50 units of EcoRI were added and the digestion was proved after incubation at 37°C for three hours by agarose gel electrophoresis.
  • the reaction was stopped by adding 1 ⁇ l of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sodium acetate and 3-4 volumes of ethanol at -80°C for 30 minutes.
  • the precipitated DNA was dissolved in 50 ⁇ l distilled water.
  • 2 ⁇ l of the linearized plasmid were mixed with 3 ⁇ l of the DNA fragment containing the methallothionein promoter and the neomycin selection gene [isolated from the plasmid pMT-neo-E (available from ATCC ) by digestion with the endonuclease EcoRI as a 4kb fragment], and ligated together. Single bacterial colonies were screened for the presence of the desired plasmid.
  • the plasmid pdhfr3.2 (available from ATCC) was digested with the restriction endonuclease HindIII. Two molecules were generated, one of 3,000 bp containing the dhfr gene sequence and one of 3,400 bp. The 3,000 bp fragment was isolated and ligated into the plasmid resulting from (5) above which was previously opened by digestion with HindIII. The resulting plasmid is represented by Fig. I-2.
  • the plasmid pUC-8-42 (available from Exogene) was digested with the restriction endonucleases EcoRI and ApaI. Two DNA molecules were generated.
  • the fragment of interest contains the U2-promoter comprising 340 bp and is easily detectable from the other fragment (3160 bp).
  • the digestion was performed in a total volume of 200 ⁇ l of reaction buffer at a final concentration of 0.5 ⁇ g/ ⁇ l DNA including 100 Units of each restriction enzyme.
  • the completion of the digest was checked after incubation at 37°C for three hours by agarose gel electrophoresis in a 0.7% agarose gel.
  • the reaction was stopped by adding 4 ⁇ l 0.5 M EDTA.
  • the 340 bp fragment was separated from the plasmid DNA by preparative 1.2% agarose gel electrophoresis.
  • the DNA was eluted from the agarose gel on DE-81 Whatman filter paper from which the DNA was removed in a high salt buffer.
  • the DNA was purified by a phenol/chloroform extraction and two ethanol precipitations.
  • the plasmid pSP165 (commercially available from Promega Biotec) containing a polylinker sequence (containing the following restriction sites: EcoRI, SacI, SmaI, AvaI, BamHI, BglII, SalI, PstI, HindIII) was linearized with the restriction enzyme EcoRI. The reaction was performed in a total volume of 50 ⁇ l and a final concentration of 1 ⁇ g/ ⁇ l plasmid DNA. 50 Units of EcoRI were added an the digestion was proved after incubation at 37°C for three hours by agarose gel electrophores.
  • the reaction was stopped by adding 1 ⁇ l of 0.5 M EDTA and the DNA was precipitated with a final concentration of 0.3 M sidium acetate and 3-4 volumes of ethanol at -80°C for 30 minutes.
  • the precipitated DNA was dissolved in 50 ⁇ l distilled water.
  • 2 ⁇ l of plasmid DNA were mixed with 10 ⁇ l of the fragment DNA containing the V2 promoter sequence, and ligated together in a total volume of 25 ⁇ l of ligation buffer containing 2 units T4-DNA ligase and 2 mM ATP at 14°C overnight. Thereafter the DNA was purified by phenol/­chloroform extractions followed by two ethanol precipitations and dissolved in 10 ⁇ l distilled water. The resulting sticky ends of EcoRI and ApaI had to be converted into blunt ends and ligated.
  • the blunt ends were converted by a removing reaction with the Mung bean nuclease as follows: to 25 ⁇ l DNA (1 ⁇ g/ ⁇ l concentration) reaction buffer, 20 units of enzyme and a final concentration of 1% glycerol to the reaction volume of 35 ⁇ l were added. After an incubation for 30 minutes at 30 C the DNA was purified by phenol/chloroform extractions followed by two ethanol precipitations. The DNA was dissolved again in 5 ⁇ l distilled water. The resulting blunt ends were ligated together in 15 ⁇ l reaction volume containing 10 x more T4 ligase then used above and 2 mM ATP at 14°C overnight.
  • the ligation mixture was added to 150 ⁇ l competent bacterial cell suspension for DNA up-take. After the DNA up-take the bacterial cells were spread on LB agar plates containing 50 ⁇ g/ml ampicillin at volumes of 50 to 300 ⁇ l cell suspension per plate. The agar plates were incubated at 37°C overnight. Single isolated bacterial colonies were screened for the presence of a plasmid containing the desired U2-promoter fragment.
  • the plasmid pBPV-342-12 (commercially available from ATCC) was digested with the endonucleases EcoRI and BamHI. Two molecules were isolated, one containing the MT promoter together with the neomycin selection gene of 4,000 bp and the plasmid of 10,000 bp.
  • the plasmid resulting from (3) above was linearized with EcoRI and ligated together with the 4,000 bp fragment containing the MP-promoter together with the neomycin selection gene. The resulting sticky ends were also converted into blunt ends and ligated together as described above.
  • the plasmid resulting from (4) above was linearized with BglII.
  • the 2.3 kb-BglII-BglII fragment was ligated together with the linearized plasmid.
  • Bacterial colonies were analysed to find the resulting plasmid.
  • the plasmid-DNA was digested with EcoRI and two resulting fragments were obtained, a 700 bp fragment (containing the promoter and a part of the HBV-sequence) and a 8,700 bp fragment (containing the rest of the HBV-sequence, MT-neo and plasmid).
  • the plasmid resulting from (5) above was digested with the endonucleases BglII and MstII. Two molecules were generated, one of 300 bp containing part of the pre-S sequence and the other (9,100 bp) which was eluted as described above. This 9,100 bp fragment was ligated to another BglII/MstII 216 bp fragment (sequence coding for an altered pre-S1 gene sequence.
  • the desired plasmid was digested with EcoRI and two resulting fragments were isolated, a 616 bp fragment and a 8,700 bp fragment.
  • the H2K promoter was isolated as an EcoRI/BglII fragment (2kb) from psp65H2 (available from Exogene).
  • the fragment containing the methallothionein promoter and the egpt-selection gene was isolated by digestion of the plasmid pMSG (available from Pharmacia) with the restriction enzyme EcoRI as a 3.6 kb fragment.
  • mammalian cells In order to achieve secretion of substantial amounts of the HBV peptides encoded by constructs of the present invention, mammalian cells must be transfected with both the construct of the present invention and a construct which will express entire S protein.
  • the cotransfection was performed in two steps (i.e., a separate transfection for each construct) or in a single step (i.e., one transfection using preparation of both constructs). Cotransfection was confirmed either by use of different selection markers on the two constructs or by detection of secretion of expression products of both constructs by immunoassay.
  • a sequence encoding the HBV peptide sequence of the present invention and a separate sequence encoding the entire S protein could be combined in a single construct.
  • restriction endonucleases used were: BglII, BamHI, HindIII, EcoRI, XbaI, MstII, XhoI, PflMI, commercially available from Gibco/BRL with their respective restriction buffers (10x).
  • restriction digests were performed and fragments were isolated as follows. Reactions typically contained 1-5 ⁇ g DNA. - distilled water was added to the DNA in an eppendorf tube to a final volume of 8 ⁇ l - 1 ⁇ l of the appropriate 10x digestion buffer was added - 1 ⁇ l (containing 5-10 U) restriction enzyme was added and mixed carefully - the reaction tube was incubated for 1 hour at 37°C - digestion was stopped by adding 0.5 M EDTA (pH 8.0) to a final concentration of 10 mM - if the DNA was analysed directly on a gel, 1 ⁇ l of gel-loading dye III (Maniatis) was added, mixed and the sample was loaded into the slots of a 0.8% agarose gel.
  • the agarose gel normally contains 0.8% agarose 1 x running buffer (TBE, Maniatis). Where a fragment (about 100-1000bp) was isolated from an agarose gel the agarose was increased to 1.2 to 1.4%.
  • the DNA to be transformed was suspended in 10 mM Tris, pH 7.5, 1 mM EDT 70 ⁇ l and added to the 200 ⁇ l bacterial cell suspension for DNA take-up.
  • the mixture was incubated on ice for 30 minutes and then 1 ml L-broth was added.
  • the mixture was incubated at 42°C for 2 minutes and at 37°C for 40 minutes.
  • the cells were spread on agar plates containing 50 ⁇ g ampicillin/ml agar at volumes of 50-300 ⁇ l cell suspension per plate.
  • the agar plates were incubated at 37°C overnight. After this incubation period, single isolated bacterial colonies were formed.
  • plasmid-bearing cells 1 liter was grown to 0.5 OD600 in L-broth and amplified for 20 hours with 200 ⁇ g/ml chloramphenicol. The culture was then centrifuged at 4,000 rpm for 20 minutes in JA-10 rotor, 4°C. The pellet was resuspended in 18 ml cold 25% sucrose, 50 mM Tris, pH 8.0, transferred to a 250 ml Erlenmeyer flask and kept on ice. 6 ml 5mg/ml lysozyme in 250 mM Tris, pH 8.0 was added and the mixture was left to stand 10-15 minutes.
  • Pronase was added to supernatant fluid to 250 ⁇ g/ml and incubated 30 minutes, 37°C.
  • the solution was extracted with phenol once with 1/2 volume phenol equilibrated with 10 mM Tris, pH 8.0, 1 mM EDTA.
  • the aqueous layer was removed.
  • Sodium acetate was then added to a final concentration of 300 mM, followed by the addition of 3 volumes cold 100% ethanol and thorough mixing. The mixture was stored at -20°C overnight.
  • the mixture was thawed and centrifuged.
  • the pellet was resuspended in 6 ml 10 mM Tris, 10 mM EDTA, pH 8.0.
  • 9.4 g CsCl and 0.65 ml of 6 mg/ml ethidium bromide were added and the volume was brought up to 10 ml with sterile double-distilled water.
  • the 10 ml alignots were put into Beckman heat-sealable gradient tubes and centrifuged, 50,000 rpm, 48 hours in Ti70.1 Beckman rotor.
  • Plasmid bands were visualized with UV and removed with syringe and 18 gauge needle by piercing the side of the tube. Ethidium bromide was removed from the plasmid fractions by 3 successive extractions with equal volumes of isobutanol. Fractions were then (1) dialyzed against one 2-liter lot of 10 mM Tris, pH 7.4, 1 mM EDTA, pH 7.5, 5 mM NaCl for 2 hours or more at 4°C; and (2) phenol extracted once with 1/3 volume phenol equilibrated as above. Sodium acetate was then added to a final concentration of 300 mM, followed by addition of two volumes of 100% ethanol. Precipitate formed at -20°C overnight, or at -70°C for 30 minutes.
  • chromosomal DNA from cell lines producing particles of this invention were isolated and digested with the appropriate restriction enzyme(s) and analysed by the method of Southern (J. Mol. Biol., Vol. 98, pp. 503-517, 1975), which is incorporated herein by reference, using a 32P-labeled DNA probe. Following digestion of the chromosomal DNA (20 ⁇ g) with the restriction enzyme BglII, the resulting fragments were separated by 0.7% agarose gel electrophoresis. Thereafter, the DNA was denatured by exposing to 366 nm UV light for 10 minutes and by incubation in a solution of 0.5 M NaOH and 1 M NaCl for 45 minutes.
  • the gels were neutralized by incubation in 0.5 M Tris, 1.5 M NaCl, pH 7.5 for 60 minutes.
  • the DNA was transferred to a nitrocellulose filter by soaking in 3 M NaCl, 0.3 M Sodiumcitrate (20 x SSC) for 20 hours through the gel by covering the top of the nitrocellulose filter with a staple of dry paper towels.
  • the nitrocellulose filter was kept for 2 hours in a vacuum oven at 80 C.
  • a radioactive DNA probe from the BglII fragment of the pHBV (2.3 kb) was prepared by nick translation.
  • the nitrocellulose filter was sealed in a plastic bag containing 10 ml of prehybridization mixture: 50% formamide, 5 x SSC, 50 mM Sodiumphosphate, pH 7.0, 5 x Denhardt's solution, 250 ⁇ g/ml denatured salmon sperm DNA.
  • the filter was incubated in this mixture for 4 hours at 45°C, after which the pre-hybridization mixture was replaced by the hybridization mixture: 50% formamide, 5 x SSC, 20 mM Sodiumphosphate, pH 7.0, 1 x Denhardt's solution, 100 ⁇ g/ml denatured salmon sperm DNA, 5 x 105 cmp/ml 32P-probe.
  • the filter after incubating in the hybridization mix for 18 hours at 45°C, was washed three times, 5 minutes each, in 0.1 x SSC, 0.1% SDS at 50°C.
  • the filter was dried at 60°C for 10 minutes and exposed to two X-ray films (XAR-5, KODAK) between two intensifying screens and kept at -80°C.
  • the first X-ray film is developed after 3 days' exposure; the second film after 7 days' exposure.
  • the recipient cells (C127 or CHO-cells available from ATCC)were seeded in normal growth medium (DMEM+10% Fetal Calf Serum, Glycose and Glutamin) into petri-dishes (1-2 x 106 cells per dish, ⁇ 10 cm) at day 1. The next day the medium was removed (4 hours before the DNA precipitate was added onto the cells), and the cells were washed twice with 1 x PBS. Then 8 ml DMEM without FCS were added. 4 hours later the DNA precipitate (prepared as described below) was added to the cells.
  • DMEM+10% Fetal Calf Serum, Glycose and Glutamin normal growth medium
  • petri-dishes 1-2 x 106 cells per dish, ⁇ 10 cm
  • Glycerol-Mix 50 ml 2 x TBS buffer, 30 ml glycerol, 120 ml distilled water
  • the Glycerol-Mix was immediately removed after an incubation at 37°C for 3 minutes and the cells were washed with 1 x PBS.
  • the cells were cultivated overnight with 8 ml of DMEM with 10% FCS.
  • the cells were recovered from the dish by treating with Trypsin-EDTA-Solution (0.025% Trypsin + 1 mM EDTA). Afterwards, to remove the Trypsin-EDTA the cells were washed with 1 x PBS, suspended in DMEM with 10% FCS and distributed into 24 costar-well-plates (cells from one dish into four 24-well-plates).
  • selection medium was added (concentration 0.5 - 1mg/ml of neomycin,or xanthine: 250 ⁇ g/ml, hypoxanthine: 15 ⁇ g/ml (or adenine: 25 ⁇ g/ml), thymidine: 10 ⁇ g/ml,aminopterine 2 ⁇ g/ml mycophenolic acid: 25 ⁇ g/ml for eco-gpt, for example).
  • the medium was changed every week. The first growing cell colonies were seen after 2 weeks.
  • TE-buffer 10 mM Trix-HCl, 1 mM EDTA, pH 7.05 was added to a final volume of 440 ⁇ l and mixed together with 60 ⁇ l 2 M CaCl2. Then the same amount of 2x TBS (Hepes 50 mM, NaCl 280 mM, Na2HPO4 1.5 mM, pH 7.05) was added and mixed well. The precipitation solution was incubated for 30 minutes at 37°C and added directly to the cells which should be transfected.
  • 2x TBS Hepes 50 mM, NaCl 280 mM, Na2HPO4 1.5 mM, pH 7.05
  • the selected cells are treated for further cultivation in normal growth medium as described in section 8.
  • Tables III - X give some of the result of ELISA analysis of immunogenic particles of the present invention as described below:
  • Table III shows the ELISA data of the purified HBs antigen particle produced from any HBV sequence construct of the present invention including the pre-S1 region with total deletion of pre-S2 and deletions upstream of the pre-S2 ATG and the S region with deletion of the S ATG and downstream the S ATG through the XBaI site (e.g. the construct of Fig. I-1) with the anti-pre-S1 monoclonal antibody MA 18/7.
  • the fractions 9-15 (Fig. XI) were pooled after CsCl sedimentation.
  • Table IV shows the ELISA data of the purified HBS antigen particle produced from any HBV sequence construct of the present invention including the pre-S1 region with total deletion of pre-S2 and deletions upstream of the pre-S2 ATG and the S region with deletion of the S ATG and downstream the S ATG through the XBaI site (e.g., the construct of Fig. I-1) with the anti-pre-S2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XI) were pooled after CsCl sedimentation.
  • Table V shows the ELISA data of the purified HBs antigen particle produced from an HBV sequence construct of the present invention including the pre-S2 region with none of the pre-S1 region and deletions upstream of the S ATG and downstream of the S ATG through the XBaI site, and the S region with deletion of the S ATG (e.g. the construct of Fig. II-1) with the anti-pre-S1 monoclonal antibody MA 18/7.
  • the fractions 9-15 (Fig. XII) were pooled after CsCl sedimentation.
  • Table VI shows the ELISA data of the purified HBS antigen particle produced from an HBV sequence construct of the present invention including the pre-S2 region with none of the pre-S1 region and deletions upstream of the S ATG and downstream of the S ATG through the XBaI site, and the S region with deletion of the S ATG (e.g. the construct of Fig. II-1) with the anti-pre-S2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XII) were pooled after CsCl sedimentation.
  • Table III CsCl-gradient ELISA Measurement Monoclonal Antibody MA 18/7 Fraction No.
  • Table VII shows the ELISA data of the purified HBs antigen particle produced from any HBV sequence construct of the present invention including the pre-S1 region with total deletion of pre-S2 and deletions upstream of the pre-S2 ATG and the S region with deletion of the S ATG (e.g., the construct of Fig. VI-2) with the anti-pre-S1 monoclonal antibody MA 18/7.
  • the fractions 9-15 (Fig. XI) were pooled after CsCl sedimentation.
  • Table VIII shows the ELISA data of the purified HBs antigen particle produced from any HBV sequence construct of the present invention including the pre-S1 region with deletions upstream of the pre-S2 ATG with deletion of the S ATG (e.g., the construct of Fig. VI-4) with the anti-pre-S2 monoclonal antibody MQ 19/10.
  • the fractions 9-15 (Fig. XI) were pooled after CsCl sedimentation.
  • Table IX shows the ELISA data of the purified HBs antigen particle produced from an HBV sequence construct of the present invention including the pre-S2 region with none of the pre-S1 region and deletions upstream of the S ATG and the S region with deletion of the S ATG (e.g., the construct of Fig. VII-2) with the anti-pre-S1 monoclonal antibody MA 18/7.
  • the fractions 9-15 (Fig. XII) were pooled after CsCl sedimentation.
  • Table X shows the ELISA data of the purified HBs antigen particle produced from an HBV sequence construct of the present invention including the pre-S2 region with deletions upstream of the S ATG with deletion of the S ATG (e.g., the construct of Fig.
  • Figure XIV shows the characterisation of the particles derived from gene constructs according to table III (Fig. I-1) and table V (Fig. II-1) cotransfected in C127 after purification in the CsCl gradient. The fraction collected had a smaller volume.
  • Table XII shows the serotyping of particles according to Fig. I-1 having the S sequence done in the Pettenkofer Institute.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
EP88109946A 1987-06-22 1988-06-22 Peptide contenant l'antigène de surface de l'hépatite B Expired - Lifetime EP0304578B1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00121167A EP1088830A3 (fr) 1987-06-22 1988-06-22 Particules de l'antigène de surface de l'hépatitie B
EP88109946A EP0304578B1 (fr) 1987-06-22 1988-06-22 Peptide contenant l'antigène de surface de l'hépatite B
LU90911C LU90911I2 (fr) 1987-06-22 2002-04-19 Antig-ne recombinant comprenant les s-quences pre-S1 et S du virus de l'h-patite B ingr-dient actif de hepacare
NL300090C NL300090I2 (nl) 1987-06-22 2002-04-19 Peptide dat het Hepatitus B-oppervlakte-antigeen omvat.

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
EP87108915 1987-06-22
EP87108914 1987-06-22
EP87108914 1987-06-22
EP87108915 1987-06-22
EP88109946A EP0304578B1 (fr) 1987-06-22 1988-06-22 Peptide contenant l'antigène de surface de l'hépatite B

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP00121167.1 Division-Into 2000-09-29

Publications (2)

Publication Number Publication Date
EP0304578A1 true EP0304578A1 (fr) 1989-03-01
EP0304578B1 EP0304578B1 (fr) 2001-10-24

Family

ID=27230073

Family Applications (2)

Application Number Title Priority Date Filing Date
EP88109946A Expired - Lifetime EP0304578B1 (fr) 1987-06-22 1988-06-22 Peptide contenant l'antigène de surface de l'hépatite B
EP00121167A Withdrawn EP1088830A3 (fr) 1987-06-22 1988-06-22 Particules de l'antigène de surface de l'hépatitie B

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP00121167A Withdrawn EP1088830A3 (fr) 1987-06-22 1988-06-22 Particules de l'antigène de surface de l'hépatitie B

Country Status (3)

Country Link
EP (2) EP0304578B1 (fr)
LU (1) LU90911I2 (fr)
NL (1) NL300090I2 (fr)

Cited By (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997008309A2 (fr) * 1995-08-29 1997-03-06 Immusol, Inc. Therapie utilisant de ribozymes contre les infections a virus de l'hepatite b
EP0761231A1 (fr) 1992-06-25 1997-03-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Composition vaccinale contenant des adjuvants
EP0811684A2 (fr) * 1991-10-09 1997-12-10 Rohto Pharmaceutical Co., Ltd. Fragments et variants du récepteur de l'oxytocine, et ADN correspondants
EP0835663A2 (fr) 1992-05-23 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins combinés contenant des antigènes de surface du virus de l'Hépatite B et d'autres antigènes
US6004561A (en) * 1993-07-30 1999-12-21 Baxter Aktiengesellschaft High level expression of polypeptide that contains modified preS1 region of hepatitis B virus large antigen
US6020167A (en) * 1990-12-19 2000-02-01 Medeva Holdings B.V. Composition used as a therapeutic agent against chronic viral hepatic diseases
US6022543A (en) * 1987-06-22 2000-02-08 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6103519A (en) * 1989-08-03 2000-08-15 Smithkline Biologicals, S.A. Antigens and methods therefor
EP1240905A1 (fr) 1996-11-07 2002-09-18 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccin acellulaire anticoquelucheux à anatoxines diphthérique et tétanique
EP1666487A1 (fr) 2000-08-10 2006-06-07 GlaxoSmithKline Biologicals SA Purification d'antigènes hbv destinés à des vaccins
WO2008028957A2 (fr) 2006-09-07 2008-03-13 Glaxosmithkline Biologicals S.A. Vaccin
US7357936B1 (en) 1998-10-16 2008-04-15 Smithkline Beecham Biologicals, Sa Adjuvant systems and vaccines
WO2008043774A1 (fr) 2006-10-12 2008-04-17 Glaxosmithkline Biologicals S.A. Vaccin
EP1925626A1 (fr) 2003-07-21 2008-05-28 Transgene S.A. Nouvelles cytokines multifonctionnelles
EP2098259A1 (fr) 2001-04-12 2009-09-09 Glaxosmithkline Biologicals S.A. Dispositif d'administration de vaccins
WO2010141861A1 (fr) 2009-06-05 2010-12-09 Infectious Disease Research Institute Adjuvants lipidiques synthétiques à base de glucopyranosyle
EP2364720A1 (fr) 2005-12-13 2011-09-14 GlaxoSmithKline Biologicals S.A. Compositions vaccinales contenant un adjuvant de saponine
WO2011130434A2 (fr) 2010-04-13 2011-10-20 Celldex Therapeutics Inc. Anticorps qui se lient au cd27 humain et utilisations de ceux-ci
EP2433648A2 (fr) 2006-10-12 2012-03-28 GlaxoSmithKline Biologicals S.A. Vaccin comprenant une émulsion adjuvante huile en l'eau
WO2012136823A1 (fr) 2011-04-08 2012-10-11 Glaxosmithkline Biologicals S.A. Procédé de fabrication d'une composition immunogène contenant du toxoïde tétanique
US8343512B2 (en) 2006-09-26 2013-01-01 Infectious Disease Research Institute Treatment of allergic conditions using a composition containing synthetic adjuvant
WO2013119856A1 (fr) 2012-02-07 2013-08-15 Infectious Disease Research Institute Formulations d'adjuvant amélioré comprenant des agonistes de tlr4 et leurs procédés d'utilisation
US8609114B2 (en) 2006-09-26 2013-12-17 Infectious Diesease Research Institute Methods of using a vaccine composition containing synthetic adjuvant
EP2682127A1 (fr) 2007-05-02 2014-01-08 GlaxoSmithKline Biologicals S.A. Vaccin
WO2014066443A1 (fr) 2012-10-23 2014-05-01 Emory University Conjugués de gm-csf et d'il-4, compositions et procédés associés
US8877208B2 (en) 2008-05-23 2014-11-04 The Regents Of The University Of Michigan Multivalent nanoemulsion vaccines
US8957047B2 (en) 2013-04-18 2015-02-17 Immune Design Corp. GLA monotherapy for use in cancer treatment
US9044420B2 (en) 2011-04-08 2015-06-02 Immune Design Corp. Immunogenic compositions and methods of using the compositions for inducing humoral and cellular immune responses
WO2015103167A2 (fr) 2013-12-31 2015-07-09 Infectious Disease Research Institute Formulations de vaccin à flacon unique
WO2016145085A2 (fr) 2015-03-09 2016-09-15 Celldex Therapeutics, Inc. Agonistes cd27
US9452209B2 (en) 2007-04-20 2016-09-27 Glaxosmithkline Biologicals Sa Influenza vaccine
US9463198B2 (en) 2013-06-04 2016-10-11 Infectious Disease Research Institute Compositions and methods for reducing or preventing metastasis
WO2017184619A2 (fr) 2016-04-18 2017-10-26 Celldex Therapeutics, Inc. Anticorps agonistes se liant au cd40 humain et leurs utilisations
WO2017200957A1 (fr) 2016-05-16 2017-11-23 Infectious Disease Research Institute Liposomes pégylés et procédés d'utilisation
WO2017200852A1 (fr) 2016-05-16 2017-11-23 Infectious Disease Research Institute Formulation contenant un agoniste de tlr et procédés d'utilisation
WO2017210364A1 (fr) 2016-06-01 2017-12-07 Infectious Disease Research Institute Nanoparticules d'alun contenant un agent d'encollage
US9895435B2 (en) 2012-05-16 2018-02-20 Immune Design Corp. Vaccines for HSV-2
WO2018232257A1 (fr) 2017-06-15 2018-12-20 Infectious Disease Research Institute Supports lipidiques nanostructurés et émulsions stables et leurs utilisations
WO2019051149A1 (fr) 2017-09-08 2019-03-14 Infectious Disease Research Institute Formulations liposomales comprenant de la saponine et procédés d'utilisation
WO2019204462A2 (fr) 2018-04-17 2019-10-24 Celldex Therapeutics, Inc. Anticorps anti-cd27 et anti-pd-l1 et constructions bispécifiques
WO2020123444A1 (fr) 2018-12-11 2020-06-18 Celldex Therapeutics, Inc. Procédés d'utilisation d'anticorps cd27 en tant que traitement de conditionnement pour une thérapie cellulaire adoptive
WO2020243115A1 (fr) 2019-05-25 2020-12-03 Infectious Disease Research Institute Composition et procédé de séchage par atomisation d'une émulsion de vaccin et d'adjuvant
WO2022051022A1 (fr) 2020-09-04 2022-03-10 Infectious Disease Research Institute Arn co-lyophilisé et support lipidique nanostructuré
WO2022217019A1 (fr) 2021-04-09 2022-10-13 Celldex Therapeutics, Inc. Anticorps contre l'anticorps ilt4, anti-ilt4/pd-l1 bispécifique et ses utilisations
WO2023091865A1 (fr) 2021-11-08 2023-05-25 Celldex Therapeutics, Inc. Constructions bispécifiques anti-ilt4 et anti-pd-1
WO2024052882A1 (fr) 2022-09-09 2024-03-14 Access To Advanced Health Institute Composition de vaccin immunogène incorporant une saponine

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1281761A1 (fr) * 2001-07-27 2003-02-05 Institut National De La Sante Et De La Recherche Medicale (Inserm) Polypeptides synthetiques derivées du proteine pre-S1 de Hepatitis B virus et utilisations associées

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0156712A1 (fr) * 1984-03-07 1985-10-02 Institut Pasteur Composition utile pour la fabrication de vaccins contenant des particules portant l'antiqène de surface du virus de l'hépatite B et le récepteur de l'albumine sérique humaine polymérisée, cellules animales capables de produire de telles particules et procédé pour leur obtention
EP0180012A1 (fr) * 1984-10-27 1986-05-07 Wolfram H. Prof. Dr. Gerlich Séquence de polypeptide immunogène du virus de l'hépatite B
WO1986005189A1 (fr) * 1985-03-06 1986-09-12 Scripps Clinic And Research Foundation Antigenes protidiques ayant des determinants dependants et independants de la conformation
EP0198474A1 (fr) * 1985-04-15 1986-10-22 SMITHKLINE BEECHAM BIOLOGICALS s.a. Antigène de surface de l'hépatite B produit par des techniques d'ADN recombinant, vaccins, diagnostics, lignées cellulaires et leur procédé de préparation
EP0244924A1 (fr) * 1986-01-31 1987-11-11 Merck & Co. Inc. Production de vaccines contre le virus de l'hépatite B
EP0248410A2 (fr) * 1986-06-03 1987-12-09 Green Cross Corporation Promoteur de levure et procédé pour préparer des protéines hétérologues
EP0250253A2 (fr) * 1986-06-20 1987-12-23 Scripps Clinic And Research Foundation Epitopes de cellules T et B de la région pré-S de l'antigène de surface de l'hépatite B

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0156712A1 (fr) * 1984-03-07 1985-10-02 Institut Pasteur Composition utile pour la fabrication de vaccins contenant des particules portant l'antiqène de surface du virus de l'hépatite B et le récepteur de l'albumine sérique humaine polymérisée, cellules animales capables de produire de telles particules et procédé pour leur obtention
EP0180012A1 (fr) * 1984-10-27 1986-05-07 Wolfram H. Prof. Dr. Gerlich Séquence de polypeptide immunogène du virus de l'hépatite B
WO1986005189A1 (fr) * 1985-03-06 1986-09-12 Scripps Clinic And Research Foundation Antigenes protidiques ayant des determinants dependants et independants de la conformation
EP0198474A1 (fr) * 1985-04-15 1986-10-22 SMITHKLINE BEECHAM BIOLOGICALS s.a. Antigène de surface de l'hépatite B produit par des techniques d'ADN recombinant, vaccins, diagnostics, lignées cellulaires et leur procédé de préparation
EP0244924A1 (fr) * 1986-01-31 1987-11-11 Merck & Co. Inc. Production de vaccines contre le virus de l'hépatite B
EP0248410A2 (fr) * 1986-06-03 1987-12-09 Green Cross Corporation Promoteur de levure et procédé pour préparer des protéines hétérologues
EP0250253A2 (fr) * 1986-06-20 1987-12-23 Scripps Clinic And Research Foundation Epitopes de cellules T et B de la région pré-S de l'antigène de surface de l'hépatite B

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF GENETIC VIROLOGY, vol. 67, 1986, pages 2305-2314, SGM, GB; H. OKAMOTO et al.: "Nucleotide sequence of a cloned hepatitis B virus genome, subtype AYR: comparison with genomes of the other three subtypes" *
JOURNAL OF IMMUNOLOGY, vol. 137, no. 8, 15th October 1986, pages 2703-2710, The American Association of Immunologists, US; D.R. MILICH et al.: "Two distinct but overlapping antibody binding sites in the pre-S(2) region of HBsAg localized within 11 continuous residues" *
JOURNAL OF IMMUNOLOGY, vol. 138, no. 12, 15th June 1987, pages 4457-4465, The American Association of Immunologists, US; D.R. MILICH et al.: "A single 10-residue pre-S(1) peptide can prime T cell help for antibody production to multiple epitopes within the pre-S(1), pre-S(2), and S regions of HBsAg1" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 81, no. 24, December 1984, pages 7708-7712, Washington, US; M.-L. MICHEL et al.: "Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin" *
SCIENCE, vol. 228, 7th June 1985, pages 1195-1199; D.R. MILICH et al.: "Enhanced immunogenicity of the pre-S region of hepatitis B surface antigen" *

Cited By (91)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6100065A (en) * 1987-06-22 2000-08-08 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6589530B1 (en) 1987-06-22 2003-07-08 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6117653A (en) * 1987-06-22 2000-09-12 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6110706A (en) * 1987-06-22 2000-08-29 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6022543A (en) * 1987-06-22 2000-02-08 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6072049A (en) * 1987-06-22 2000-06-06 Medeva Holdings B.V. Hepatitis B surface antigen vaccine
US6103519A (en) * 1989-08-03 2000-08-15 Smithkline Biologicals, S.A. Antigens and methods therefor
US6020167A (en) * 1990-12-19 2000-02-01 Medeva Holdings B.V. Composition used as a therapeutic agent against chronic viral hepatic diseases
EP0811684A2 (fr) * 1991-10-09 1997-12-10 Rohto Pharmaceutical Co., Ltd. Fragments et variants du récepteur de l'oxytocine, et ADN correspondants
EP0811684A3 (fr) * 1991-10-09 1998-01-07 Rohto Pharmaceutical Co., Ltd. Fragments et variants du récepteur de l'oxytocine, et ADN correspondants
EP2156845A1 (fr) 1992-05-23 2010-02-24 GlaxoSmithKline Biologicals SA Vaccins combinés contenant des antigènes de surface du virus de l'Hépatite B et d'autres antigènes
EP0835663A2 (fr) 1992-05-23 1998-04-15 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins combinés contenant des antigènes de surface du virus de l'Hépatite B et d'autres antigènes
EP2289548A2 (fr) 1992-05-23 2011-03-02 GlaxoSmithKline Biologicals S.A. Vaccins combinés contenant des antigènes de surface du virus de l'hepatite B et d'autres antigènes
EP0761231A1 (fr) 1992-06-25 1997-03-12 SMITHKLINE BEECHAM BIOLOGICALS s.a. Composition vaccinale contenant des adjuvants
US6004561A (en) * 1993-07-30 1999-12-21 Baxter Aktiengesellschaft High level expression of polypeptide that contains modified preS1 region of hepatitis B virus large antigen
WO1997008309A2 (fr) * 1995-08-29 1997-03-06 Immusol, Inc. Therapie utilisant de ribozymes contre les infections a virus de l'hepatite b
WO1997008309A3 (fr) * 1995-08-29 1997-07-10 Immusol Inc Therapie utilisant de ribozymes contre les infections a virus de l'hepatite b
EP1240905A1 (fr) 1996-11-07 2002-09-18 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccin acellulaire anticoquelucheux à anatoxines diphthérique et tétanique
EP2266604A2 (fr) 1998-10-16 2010-12-29 GlaxoSmithKline Biologicals S.A. Produits adjuvants et vaccins
US7357936B1 (en) 1998-10-16 2008-04-15 Smithkline Beecham Biologicals, Sa Adjuvant systems and vaccines
US8628784B2 (en) 1998-10-16 2014-01-14 Glaxosmithkline Biologicals S.A. Adjuvant systems and vaccines
EP1666487A1 (fr) 2000-08-10 2006-06-07 GlaxoSmithKline Biologicals SA Purification d'antigènes hbv destinés à des vaccins
BG66038B1 (bg) * 2000-08-10 2010-11-30 Glaxo Smithkline Biologicals S.A. Пречистване на hbv антигени за използване във ваксини
EP2098259A1 (fr) 2001-04-12 2009-09-09 Glaxosmithkline Biologicals S.A. Dispositif d'administration de vaccins
EP1925626A1 (fr) 2003-07-21 2008-05-28 Transgene S.A. Nouvelles cytokines multifonctionnelles
EP1944318A1 (fr) 2003-07-21 2008-07-16 Transgene S.A. Nouvelles cytokines multifonctionnelles
EP2364720A1 (fr) 2005-12-13 2011-09-14 GlaxoSmithKline Biologicals S.A. Compositions vaccinales contenant un adjuvant de saponine
US10039823B2 (en) 2005-12-13 2018-08-07 Glaxosmithkline Biologicals, S.A. Vaccine compositions comprising a saponin adjuvant
US10143745B2 (en) 2005-12-13 2018-12-04 GlacoSmithKline Biologicals, S.A. Vaccine compositions comprising a saponin adjuvant
EP2364722A1 (fr) 2005-12-13 2011-09-14 GlaxoSmithKline Biologicals S.A. Compositions vaccinales contenant un adjuvant de saponine
EP2364721A1 (fr) 2005-12-13 2011-09-14 GlaxoSmithKline Biologicals S.A. Compositions vaccinales contenant un adjuvant de saponine
EP2364723A1 (fr) 2005-12-13 2011-09-14 GlaxoSmithKline Biologicals S.A. Compositions vaccinales contenant un adjuvant de saponine
EP2364724A1 (fr) 2005-12-13 2011-09-14 GlaxoSmithKline Biologicals S.A. Compositions vaccinales contenant un adjuvant de saponine
WO2008028956A1 (fr) 2006-09-07 2008-03-13 Glaxosmithkline Biologicals S.A. Vaccin
WO2008028957A2 (fr) 2006-09-07 2008-03-13 Glaxosmithkline Biologicals S.A. Vaccin
US9987355B2 (en) 2006-09-26 2018-06-05 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
US10792359B2 (en) 2006-09-26 2020-10-06 Infectious Disease Research Institute Methods of using a vaccine composition containing synthetic adjuvant
US8343512B2 (en) 2006-09-26 2013-01-01 Infectious Disease Research Institute Treatment of allergic conditions using a composition containing synthetic adjuvant
US9907845B2 (en) 2006-09-26 2018-03-06 Infectious Disease Research Institute Methods of using a vaccine composition containing synthetic adjuvant
US8609114B2 (en) 2006-09-26 2013-12-17 Infectious Diesease Research Institute Methods of using a vaccine composition containing synthetic adjuvant
US9950063B2 (en) 2006-09-26 2018-04-24 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
US10765736B2 (en) 2006-09-26 2020-09-08 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
EP3795173A1 (fr) 2006-09-26 2021-03-24 Infectious Disease Research Institute Composition de vaccin contenant un adjuvant synthétique
US8840908B2 (en) 2006-09-26 2014-09-23 Infectious Disease Research Institute Vaccine composition containing synthetic adjuvant
WO2008043774A1 (fr) 2006-10-12 2008-04-17 Glaxosmithkline Biologicals S.A. Vaccin
EP2433648A2 (fr) 2006-10-12 2012-03-28 GlaxoSmithKline Biologicals S.A. Vaccin comprenant une émulsion adjuvante huile en l'eau
US9700605B2 (en) 2006-10-12 2017-07-11 Glaxosmithkline Biologicals S.A. Vaccine comprising an oil in water emulsion
US9597389B2 (en) 2007-04-20 2017-03-21 Glaxosmithkline Biologicals Sa Oil-in-water emulsion influenza vaccine
US10548969B2 (en) 2007-04-20 2020-02-04 Glaxosmithkline Biologicals Sa Oil-in-water emulsion influenza vaccine
US10016495B2 (en) 2007-04-20 2018-07-10 Glaxosmithkline Biologicals S.A. Oil-in-water emulsion influenza vaccine
US9452209B2 (en) 2007-04-20 2016-09-27 Glaxosmithkline Biologicals Sa Influenza vaccine
EP2682127A1 (fr) 2007-05-02 2014-01-08 GlaxoSmithKline Biologicals S.A. Vaccin
EP3251680A1 (fr) 2008-05-22 2017-12-06 Infectious Disease Research Institute Composition de vaccin contenant un adjuvant synthétique
US9415006B2 (en) 2008-05-23 2016-08-16 The Regents Of The University Of Michigan Immunogenic compositions comprising nanoemulsion and hepatitis B virus immunogen and methods of using the same
US8877208B2 (en) 2008-05-23 2014-11-04 The Regents Of The University Of Michigan Multivalent nanoemulsion vaccines
EP3124491A1 (fr) 2009-06-05 2017-02-01 Infectious Disease Research Institute Adjuvants lipidiques synthétiques à base de glucopyranosyle et compositions de vaccin les contenant
WO2010141861A1 (fr) 2009-06-05 2010-12-09 Infectious Disease Research Institute Adjuvants lipidiques synthétiques à base de glucopyranosyle
EP3165540A1 (fr) 2010-04-13 2017-05-10 Celldex Therapeutics, Inc. Anticorps liant un cd27 humain et leurs utilisations
WO2011130434A2 (fr) 2010-04-13 2011-10-20 Celldex Therapeutics Inc. Anticorps qui se lient au cd27 humain et utilisations de ceux-ci
WO2012136823A1 (fr) 2011-04-08 2012-10-11 Glaxosmithkline Biologicals S.A. Procédé de fabrication d'une composition immunogène contenant du toxoïde tétanique
US9044420B2 (en) 2011-04-08 2015-06-02 Immune Design Corp. Immunogenic compositions and methods of using the compositions for inducing humoral and cellular immune responses
EP3563834A1 (fr) 2012-02-07 2019-11-06 Infectious Disease Research Institute Formulations d'adjuvant amélioré comprenant des agonistes de tlr4 et leurs procédés d'utilisation
WO2013119856A1 (fr) 2012-02-07 2013-08-15 Infectious Disease Research Institute Formulations d'adjuvant amélioré comprenant des agonistes de tlr4 et leurs procédés d'utilisation
US9895435B2 (en) 2012-05-16 2018-02-20 Immune Design Corp. Vaccines for HSV-2
EP3587455A1 (fr) 2012-10-23 2020-01-01 Emory University Conjugués de gm-csf et d'il-4, compositions et procédés associés
WO2014066443A1 (fr) 2012-10-23 2014-05-01 Emory University Conjugués de gm-csf et d'il-4, compositions et procédés associés
US8962593B2 (en) 2013-04-18 2015-02-24 Immune Design Corp. GLA monotherapy for use in cancer treatment
US10342815B2 (en) 2013-04-18 2019-07-09 Immune Design Corp. GLA monotherapy for use in cancer treatment
US8957047B2 (en) 2013-04-18 2015-02-17 Immune Design Corp. GLA monotherapy for use in cancer treatment
US10993956B2 (en) 2013-04-18 2021-05-04 Immune Design Corp. GLA monotherapy for use in cancer treatment
US9463198B2 (en) 2013-06-04 2016-10-11 Infectious Disease Research Institute Compositions and methods for reducing or preventing metastasis
WO2015103167A2 (fr) 2013-12-31 2015-07-09 Infectious Disease Research Institute Formulations de vaccin à flacon unique
EP3915579A1 (fr) 2013-12-31 2021-12-01 Infectious Disease Research Institute Formulations de vaccin à flacon unique
US11801223B2 (en) 2013-12-31 2023-10-31 Access To Advanced Health Institute Single vial vaccine formulations
WO2016145085A2 (fr) 2015-03-09 2016-09-15 Celldex Therapeutics, Inc. Agonistes cd27
WO2017184619A2 (fr) 2016-04-18 2017-10-26 Celldex Therapeutics, Inc. Anticorps agonistes se liant au cd40 humain et leurs utilisations
EP4112638A1 (fr) 2016-05-16 2023-01-04 Access to Advanced Health Institute Formulation contenant un agoniste de tlr et procédés d'utilisation
WO2017200852A1 (fr) 2016-05-16 2017-11-23 Infectious Disease Research Institute Formulation contenant un agoniste de tlr et procédés d'utilisation
US11266602B2 (en) 2016-05-16 2022-03-08 Infectious Disease Research Institute PEGylated liposomes and methods of use
WO2017200957A1 (fr) 2016-05-16 2017-11-23 Infectious Disease Research Institute Liposomes pégylés et procédés d'utilisation
WO2017210364A1 (fr) 2016-06-01 2017-12-07 Infectious Disease Research Institute Nanoparticules d'alun contenant un agent d'encollage
WO2018232257A1 (fr) 2017-06-15 2018-12-20 Infectious Disease Research Institute Supports lipidiques nanostructurés et émulsions stables et leurs utilisations
US11141377B2 (en) 2017-06-15 2021-10-12 Infectious Disease Research Institute Nanostructured lipid carriers and stable emulsions and uses thereof
WO2019051149A1 (fr) 2017-09-08 2019-03-14 Infectious Disease Research Institute Formulations liposomales comprenant de la saponine et procédés d'utilisation
WO2019204462A2 (fr) 2018-04-17 2019-10-24 Celldex Therapeutics, Inc. Anticorps anti-cd27 et anti-pd-l1 et constructions bispécifiques
WO2020123444A1 (fr) 2018-12-11 2020-06-18 Celldex Therapeutics, Inc. Procédés d'utilisation d'anticorps cd27 en tant que traitement de conditionnement pour une thérapie cellulaire adoptive
WO2020243115A1 (fr) 2019-05-25 2020-12-03 Infectious Disease Research Institute Composition et procédé de séchage par atomisation d'une émulsion de vaccin et d'adjuvant
WO2022051022A1 (fr) 2020-09-04 2022-03-10 Infectious Disease Research Institute Arn co-lyophilisé et support lipidique nanostructuré
WO2022217019A1 (fr) 2021-04-09 2022-10-13 Celldex Therapeutics, Inc. Anticorps contre l'anticorps ilt4, anti-ilt4/pd-l1 bispécifique et ses utilisations
WO2023091865A1 (fr) 2021-11-08 2023-05-25 Celldex Therapeutics, Inc. Constructions bispécifiques anti-ilt4 et anti-pd-1
WO2024052882A1 (fr) 2022-09-09 2024-03-14 Access To Advanced Health Institute Composition de vaccin immunogène incorporant une saponine

Also Published As

Publication number Publication date
NL300090I1 (nl) 2002-07-01
LU90911I2 (fr) 2002-06-19
EP1088830A2 (fr) 2001-04-04
EP0304578B1 (fr) 2001-10-24
EP1088830A3 (fr) 2004-04-07
NL300090I2 (nl) 2002-12-02

Similar Documents

Publication Publication Date Title
EP0304578B1 (fr) Peptide contenant l'antigène de surface de l'hépatite B
US6110706A (en) Hepatitis B surface antigen vaccine
US6306625B1 (en) Method for obtaining expression of mixed polypeptide particles in yeast
Tiollais et al. Biology of hepatitis B virus
JP2634371B2 (ja) 脊椎動物細胞内でｂ型肝炎表面抗原をコードするｄｎａを発現するベクター
EP0421635B1 (fr) Protéines chimériqués d'antigène du noyau d'Hepadnavirus
EP0563093B2 (fr) Composition utilisee comme agent therapeutique contre des maladies hepatiques virales chroniques
US6544757B1 (en) Synthesis of human virus antigens by yeast
US5098704A (en) Hepatitis surface antigen particle vaccine
JPH0690769A (ja) バキュロウイルス発現系
US5324513A (en) Composition useful for the fabrication of vaccines
CA1222707A (fr) Preparation d'un vaccin contre l'hepatite b
CA1341021C (fr) Vaccin contre l'hepatite comprenant des particules antigeniques de surface
AU619753B2 (en) Hepatitis b surface antigen vaccine
EP0241021A2 (fr) Procédé de production de HBsAg contenant la séquence d'acides aminés codée par la région pre S tardive du HB virus et ce HBsAg
AU625348B2 (en) Heterologous viral peptide particle immunogens
KR960004264B1 (ko) B형 간염 표면항원, 이의 생성 진핵 세포 및 백신의 제조방법, 검정방법 및 진단용 키트

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE ES FR GB GR IT LI LU NL SE

17P Request for examination filed

Effective date: 19890901

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: EPITEC LIMITED

RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: EPITEC LIMITED

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: MEDEVA HOLDINGS BV

17Q First examination report despatched

Effective date: 19951006

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE CH DE ES FR GB GR IT LI LU NL SE

REF Corresponds to:

Ref document number: 207535

Country of ref document: AT

Date of ref document: 20011115

Kind code of ref document: T

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REF Corresponds to:

Ref document number: 3856496

Country of ref document: DE

Date of ref document: 20011129

REG Reference to a national code

Ref country code: GB

Ref legal event code: IF02

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: PATENTANWAELTE SCHAAD, BALASS, MENZL & PARTNER AG

ET Fr: translation filed
REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 88109946

Country of ref document: AT

Kind code of ref document: T

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20010402323

Country of ref document: GR

REG Reference to a national code

Ref country code: FR

Ref legal event code: CP

Free format text: PRODUCT NAME: ANTIGENES DE SURFACE RECOMBINANTS DE L'HEPATITE B (S,PRE-S1, PRE-S2); REGISTRATION NO/DATE: EU/1/00/136/001 20000804

Spc suppl protection certif: 02C0017

Filing date: 20020424

REG Reference to a national code

Ref country code: SE

Ref legal event code: SPCF

Free format text: 0290007-4, 20000804

REG Reference to a national code

Ref country code: GB

Ref legal event code: CTFF

Free format text: GBCTFFSPC/GB02/020: 20020422

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2167304

Country of ref document: ES

Kind code of ref document: T3

REG Reference to a national code

Ref country code: LU

Ref legal event code: CCP

Free format text: LUCCP 90911, EXPIRES: 20130622

REG Reference to a national code

Ref country code: NL

Ref legal event code: AC1

Free format text: NLAC1 300090, 20020419

REG Reference to a national code

Ref country code: AT

Ref legal event code: ESZA

Ref document number: 88109946

Country of ref document: AT

Kind code of ref document: T

Free format text: PRODUCT NAME: NICHT NATUERLICHES PRAE-S1-S-PROTEIN VOM UNTERTYP AYW IN DER PRAE-S1-REGION UND VOM UNTERTYP ADW IN DER S-REGION HERGESTELLT IN C127-MAUSZELLEN

Spc suppl protection certif: SZ 13/2002

Filing date: 20020424

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed
REG Reference to a national code

Ref country code: GB

Ref legal event code: CTFG

Spc suppl protection certif: SPC/GB02/020

Filing date: 20030313

Expiry date: 20130612

REG Reference to a national code

Ref country code: SE

Ref legal event code: SPCG

Spc suppl protection certif: 02900074.L

Ref country code: SE

Ref legal event code: SPCG

Free format text: 0290007-4

Spc suppl protection certif: 0290007-4

REG Reference to a national code

Ref country code: FR

Ref legal event code: CR

Free format text: 02C0017, 20020424

Spc suppl protection certif: 02C0017

Filing date: 20020424

REG Reference to a national code

Ref country code: FR

Ref legal event code: CP

REG Reference to a national code

Ref country code: AT

Ref legal event code: EEZF

Ref document number: 207535

Country of ref document: AT

Kind code of ref document: T

Free format text: PRODUCT NAME: NICHT NATUERLICHES PRAE-S1-S-PROTEIN VOM UNTERTYP AYW IN DER PRAE-S1-REGION UND VOM UNTERTYP ADW IN DER S-REGION HERGESTELLT IN C127-MAUSZELLEN

Spc suppl protection certif: SZ 13/2002

Filing date: 20020424

Extension date: 20130622

Effective date: 20060607

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20070603

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20070607

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DE

Payment date: 20070614

Year of fee payment: 20

Ref country code: AT

Payment date: 20070614

Year of fee payment: 20

Ref country code: CH

Payment date: 20070614

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: LU

Payment date: 20070705

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: ES

Payment date: 20070717

Year of fee payment: 20

REG Reference to a national code

Ref country code: FR

Ref legal event code: CY

Free format text: PRODUCT NAME: ANTIGENE DE SURFACE RECOMBINANT DE L?HEPATITE B (PRE-S1); REGISTRATION NO/DATE IN FRANCE: EU/1/00/136/001 DU 20000804; REGISTRATION NO/DATE AT EEC: EU/1/00/136/001 DU 20000804

Spc suppl protection certif: 02C0017

Filing date: 20020424

Extension date: 20130621

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20070620

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: IT

Payment date: 20070625

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: BE

Payment date: 20070816

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20070608

Year of fee payment: 20

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GR

Payment date: 20070514

Year of fee payment: 20

BE20 Be: patent expired

Owner name: *MEDEVA HOLDINGS B.V.

Effective date: 20080622

REG Reference to a national code

Ref country code: GB

Ref legal event code: PE20

Expiry date: 20080621

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

EUG Se: european patent has lapsed
NLV7 Nl: ceased due to reaching the maximum lifetime of a patent

Effective date: 20080622

REG Reference to a national code

Ref country code: AT

Ref legal event code: EELA

Ref document number: 207535

Country of ref document: AT

Kind code of ref document: T

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20080622

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20080621

REG Reference to a national code

Ref country code: BE

Ref legal event code: CCRE

Free format text: PRODUCT NAME: TRIPLE ANTIGEN HEPATITIS B VACCINE (RECOMBINANT); REGISTRATION NO/DATE: EU/1/00/136/001 20000804

Spc suppl protection certif: 2002C/010

Filing date: 20020419

Expiry date: 20080622

Extension date: 20130622

Effective date: 20080630

REG Reference to a national code

Ref country code: GB

Ref legal event code: CTFL

Free format text: PRODUCT NAME: RECOMBINANT ANTIGEN COMPRISING PRE-S1 AND S SEQUENCE OF HEPATITIS B VIRUS; REG.NO/DATE: EU/1/00/136/001-002 20000804

Spc suppl protection certif: SPC/GB02/020

Filing date: 20020422

Effective date: 20080622

REG Reference to a national code

Ref country code: ES

Ref legal event code: FD2A

Effective date: 20120510

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF EXPIRATION OF PROTECTION

Effective date: 20080623

REG Reference to a national code

Ref country code: GB

Ref legal event code: SPCX

Free format text: SPC/GB02/020 IS INCORRECTLY SHOWING AS SURRENDERED, BUT THEY SHOULD BE SHOWING AS FEES NOT PAID IN THE REASON FOR LAPSE SECTION.

Spc suppl protection certif: SPC/GB02/020